Ilyinskii P O, Desrosiers R C
New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102, USA.
J Virol. 1996 May;70(5):3118-26. doi: 10.1128/JVI.70.5.3118-3126.1996.
Ten mutants of the simian immunodeficiency virus (SIV) SIVmac239 bearing deletions (delta) or substitutions (subst) in the NF-kappaB and/or Sp1 binding elements were created, and the replicative capacities of the mutants were analyzed. All mutants, including one extensively mutagenized strain entirely missing the NF-kappaB and four Spl binding elements, replicated with wild-type kinetics and to a wild-type level in peripheral blood mononuclear cell cultures in 50 to 100% of the experiments. One group of mutants replicated very similarly to SIVmac239 in kinetics and yield in CEMxl74 cells (2xNFKappaB > or = SlVmac239 approximately deltaNFkappaB approximately deltaSpl234 approximately substNFkappaB approximately substSpl2 approximately substSp23), while a second group replicated with delayed or slightly delayed kinetics in CEMxl74 cells (SIVmac239 > substSp34 > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSp1 > substSpl234). Reversions or additional mutations were not detected in the U3 and R regions of proviral DNA from CEMxl74 cells infected with the SIVmac239 mutants. Similar results were obtained when mutants of SIVmacMER (a macrophage-competent derivative of SIVmac239) were tested in peripheral blood mononuclear cell and CEMx174 cultures. However, the growth of most mutated viruses was suppressed in primary rhesus monkey alveolar macrophages (SIVmacMER approximately 2xNFkappaB approximately substNFkappaB > deltaNFkappaB > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSpl > deltaSpl234 approximately substSpl2 > substSp23 approximately substSp34 approximately substSpl234 > or = SIVmac239). Thus, changes in the Sp1 binding sites had the most dramatic effects on SIVmac replication in primary macrophage cultures. Analysis of long terminal repeat-driven secreted alkaline phosphatase activity in transient assays showed that, unlike human immunodeficiency virus type 1, the SIV long terminal repeat possesses an enhancer region just upstream of the NF-kappaB element which maintains significant levels of basal transcription in the absence of NF-kappaB and Sp1 sites. This region is responsive to transactivation by Tat. In addition, the SIV TATA box was shown to be stronger than that of human immunodeficiency virus type 1. Therefore, the surprisingly high replicative capacity of NF-kappaB and Sp1 binding site mutants of SIVmac is due to unique features or the enhancer/promoter region.
构建了10个在核因子κB（NF-κB）和/或Sp1结合元件上带有缺失（Δ）或替换（subst）的猿猴免疫缺陷病毒（SIV）SIVmac239突变体，并分析了这些突变体的复制能力。所有突变体，包括一个完全缺失NF-κB和4个Sp1结合元件的广泛诱变菌株，在50%至100%的实验中，在外周血单核细胞培养物中以野生型动力学进行复制，且复制水平达到野生型。一组突变体在CEMx174细胞中的动力学和产量与SIVmac239非常相似（2xNFκB≥SIVmac239≈ΔNFκB≈ΔSp1234≈substNFκB≈substSp12≈substSp23），而另一组在CEMx174细胞中的复制动力学延迟或略有延迟（SIVmac239＞substSp34＞ΔNFκBΔSp1234≈ΔNFκBΔSp1＞substSp1234）。在感染SIVmac239突变体的CEMx174细胞的原病毒DNA的U3和R区域未检测到回复突变或额外突变。当在SIVmacMER（SIVmac239具有巨噬细胞感染能力的衍生物）的突变体在外周血单核细胞和CEMx174培养物中进行测试时，获得了类似的结果。然而，大多数突变病毒在原代恒河猴肺泡巨噬细胞中的生长受到抑制（SIVmacMER≈2xNFκB≈substNFκB＞ΔNFκB＞ΔNFκBΔSp1234≈ΔNFκBΔSp1＞ΔSp1234≈substSp12＞substSp23≈substSp34≈substSp1234≥SIVmac239）。因此，Sp1结合位点的变化对原代巨噬细胞培养物中SIVmac的复制影响最为显著。在瞬时分析中对长末端重复序列驱动的分泌碱性磷酸酶活性的分析表明，与1型人类免疫缺陷病毒不同，SIV长末端重复序列在NF-κB元件上游具有一个增强子区域，该区域在没有NF-κB和Sp1位点的情况下维持显著水平的基础转录。该区域对Tat的反式激活有反应。此外，SIV的TATA框比1型人类免疫缺陷病毒的更强。因此，SIVmac的NF-κB和Sp1结合位点突变体具有惊人的高复制能力是由于增强子/启动子区域具有独特特征。
